Agriculture. The present invention refers to the obtaining of regulatory (xe2x80x9cpromoterxe2x80x9d) DNA sequences and the construction of new chimeric genes, and to the use of these sequences, capable of being specifically expressed in transgenic plant seeds. Ha ds10 G1 gene has the peculiarity of only being expressed in sunflower seeds from the maturation until the desiccation phase, without responding to hormones such as abscicic acid (ABA) or water stress in vegetative tissues. Furthermore, gene Ha ds10 G1 is expressed homogeneously in immature embryos and preferably in the palisade parenchyma of mature embryo cotyledons. These expression patterns, as well as the high activity levels of the gene, suggest that its regulatory sequences are particularly appropriate for the genetic manipulation of storage substances in seeds.
Up to now in order to confer specific expression in transgenic plant seeds, promoters have been isolated, characterised and used, especially belonging to plant genes which code for storage proteins or other products solely expressed in seeds, during different phases of development [see the following references and patents, as well as other documents cited therein Thomas T L, in Plant Cell, vol 5, pp 1401-1410, 1993; Gatehouse J A and Shirsat A H in Control of Plant Gene Expression, pp 357-375, CRC press, 1993; U.S. Pat. Nos. 5,530,192, 5,530,194 and 5,420,034]. For example, this has allowed the obtaining of new transgenic plants with modified fatty acid and storage protein content [see: Voelker T A, Worrell A C, Anderson L, Bleibaum 3, Fan C, Hawkins D J, Radke S E and Davies H M, in Science, vol. 257, pp.72-74, 1992; and Saalbach I, Pickardt T, Machemehl F, Saalbach G, Schieder O, and Muntz K, in Molecular and General Genetics 242: 226-236, 1994]. Other promoters with different tissue specificity in seed and varied temporal expression patterns could be useful for the development of the enormous potential of this technique. Recently the expression in seeds of genes that code for low molecular weight heat shock proteins was described (sHSPs: small heat-shock proteins). One of these genes, Ha hsp17.7 G4, shows in tobacco transgenic plants, expression patterns appropriate for its possible use in the genetically engineered modification of seeds: this gene is expressed from early seed maturation phases, and is specific of cotyledon tissue [Coca M A, Almoguera C, Thomas T L and Jordano J, in: Plant Molecular Biology 31: 863-876, 1996]. However, gene Ha hsp17.7 G4, like other sHSP plant genes expressed in seeds, is also expressed in response to heat (heat shock) in plant vegetative tissues after seed germination. The latter makes its use in genetic engineering impossible in the case that regulatory DNA sequences that guarantee the absence of expression of chimeric genes outside of the seed are required: for example, when the expression elsewhere of these genes may affect the viability, the growth, or the health of the transgenic plants. To solve these problems the Ha hsp17.7 G4 gene regulatory sequences were modified in such a way that the chimeric genes containing these sequences maintain their expression in seeds, and lose their heat induction; a procedure which can be used for the modification and similar use of regulatory sequences of other sHSP genes expressed in seed [Almoguera, Prieto-Dapena and Jordano, patent request #9602746 (Spanish Patent Office)]. Alternatively, a similar use for the promoter and regulatory sequences of the sunflower gene Ha hsp17.6 G1, which is only expressed in seeds, is proposed. This gene does not respond to heat or other types of stress (cold, dehydration, ABA hormone treatment) in vegetative tissues [Carranco, Almoguera and Jordano, Spanish patent application #970121.
Alternative analogous uses for promoter and regulatory sequences of sunflower LEA Ha ds10 G1 gene are proposed. Gene Ha ds10 G1 has been found in a genomic clone corresponding to a previously described cDNA (Ha ds10, access number X506999) whose expression patterns were not totally known [Almoguera and Jordano, Plant Mol. Biol. 19:781-792, 1992]. The promoter and regulatory sequences of this gene (Ha ds10 G1) have been cloned and are described, characterised and used for the first time in the examples of this application. The Ha ds10 G1 gene belongs to the Class I LEA (Late Embryogenesis Abundant) gene family (D-19 or LEA-I type ) These genes code for highly conserved proteins in various plant species, and their expression is usually restricted to seeds and early germination phases [see for example the following reviews: Dure III, L., Structural motifs in Lea proteins, in Plant Responses to Plant Dehydration During Environmental Stress., Close T J and Bray E A Eds., Current Topics in Plant Physiology 10: 91-103, 1993; and Delseny M, Gaubier P, Hull G. Saez-Vasquez J, Gallois P, Raynal M, Cooke R, Grellet F., Nuclear Genes expressed during seed desiccation: relationship with responses to stress, in Stress-induced Gene Expression in Plants (Basra, A. S., ed.), pp. 25-59, Harwood Academic Publishers, Reading, 1994]. LEA gene promoters have not been considered so good candidates for their use in seed storage substance modification projects, since usually their activity is expressed in later seed maturation phases, such as embryo desiccation [Kridls J C, Knauf V C, Thompson G a in Control of Plant Gene Expression. pp. 481-498, CRC press, 1993]. However, LEA genes that are activated in maturation phases prior to desiccation are known, such as the cotton genes denominated LEA-A [Hughes D W and Galau G A, The Plant Cell 3:605-618, 1991]. Examples of activation prior to desiccation are also known in the class I LEA genes, such as in the case of At Em1, emb564 and emb1 genes [in arabidopsis, maize and carrot, respectively: Gaubier P, Raynal M, Hull G, Huestis G M, Grellet F, Arenas C, Pages M, and Delseny M, Mol. Gen. Genet., 238: 409-418, 1993; Williams B, and Tsang A, Plant Mol. Biol., 16: 919-923, 1991; Wurtele E S, Wang H, Durgerian S, Nikolau B J, and Ulrich T H. Plant Physiol. 102:303-312, 1993]. These examples seem to indicate the possible use of regulatory sequences from genes of this family, for the modification of seeds. However, its specific use would be limited both by the expression levels obtained in each case and in each development phase; as well as by the different tissue specificity. Thus, even though in Arabidopsis the At Em1 gene is early activated, its expression is basically restricted to cotyledon provascular tissue and cortical tissue external to the embryonic axis [Gaubier, P., Raynal, M., Hull, G., Huestis, G M., Grellet, F., Arenas, C., Pages, M., and Delseny, M., Mol. Gen. Genet., 238: 409-418, 1993]. In the case of the carrot gene, emb1, its mRNA are preferably localised in the embryonic meristems, especially in the procambium [Wurtele E S, Wang H, Durgerian S, Nikolau B J, and Lylrich T H. Plant Physiol. 102:303-312, 1993]. No gene sequence of the emb564 gene has been published and the exact localisation of its mRNA is unknown [Williams B and Tsang A, Plant Mol. Biol., 16: 919-923, 1991].
The expression of sunflower gene Ha ds10 G1, as well as its promoter and regulatory sequences present unique characteristics among the other members of the LEA-I family, as described below, which means that these sequences may be potentially used for the modification of seeds by genetic engineering.
The present invention refers to the isolation and characterisation of the promoter and regulatory sequences of a sunflower LEA-I gene, Ha ds10 G1, in transgenic tobacco plants. These sequences (Example 1) present highly appropriate characteristics for their use in the modification of seeds (e.g. storage substances). The advantages of their possible use in transgenic plants are demonstrated in other examples: A. Studies of HA ds10 mRNA accumulation and localisation in the homologous system (Example 2). These studies demonstrate both the high expression levels reached during embryogenesis since early maturation phases, as well as the absolute seed specific localisation, accompanied of a homogenous distribution in embryos, which terminates essentially restricted to the cotyledon palisade parenchyma, a tissue specialised in the accumulation of sunflower storage substances. B. In example 3 the possible use of such sequences, via the construction and analysis of various chimeric genes in transgenic plants, is illustrated, using the promoter and combinations of various Ha ds10 G1 regulatory sequences (5xe2x80x2-flanking, coding, intron and 3xe2x80x2-flanking), with the reporter gene of bacterial xcex2-glucuronidase (GUS). These examples demonstrate the usefulness of the different chimeric genes tested in a heterologous model (tobacco): high expression level and seed specificity since early maturation phases, as well as the functional contribution of the various sequences tested. The examples attached demonstrate that the seed specificity is basically conferred by the promoter and the 5xe2x80x2-flanking sequences of Ha ds10G1 (including untranscribed and transcribed sequences, such as the 5xe2x80x2-UTR, and part of the coding sequence). Additionally, the 3xe2x80x2-flanking sequences increase expression levels in seeds, and the intron specifically reduces the expression levels in non-embryonic tissues. Due to the conservation of the regulation of embryonic gene expression in plant seeds, including LEA-I genes [Thomas T L, in I 5:1401-1410, 1993]; these sequences could be used both in the homologous system (sunflower) as in other heterologous systems of great economic importance (for example oilseed rape, soybean, maize, etc).
The practical embodiment of this invention, represented by the attached examples and figures, uses conventional Molecular Biology, Microbiology, recombinant DNA and transgenic plant production techniques that are common practice in laboratories specialised in these fields. These techniques have been explained in sufficient detail in the scientific literature [Sambrok J, Fritsch E F, and Maniatis T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory Press, 2nd Edition, 1989; Glover D M, DNA Cloning, IRL Press, 1985; Lindsey K., Plant Tissue Culture Manual, Kluwer Academic Publishers, 1993; and Gelvin S B, Schilperoort R A, Verma D P S, Plant Molecular Biology Manual, Kluwer Academic Publishers, 1992]. For more details, other references are cited in the corresponding section in this application.